The p53 transcription factor plays a pivotal role in tumorigenesis. p53 inactivation in tumors, which occurs typically through missense mutation, unequivocally promotes cancer, but the mutated p53 protein also displays gain-of-function (GOF) properties that promote cancer. In this proposal, we strive to deconstruct the transcriptional programs through which wild-type p53 suppresses cancer and through which missense mutant p53 exerts GOF effects to promote cancer. We propose to use integrated genetic, genomic, cell biological and biochemical approaches to define the p53 transcriptional programs critical for p53-mediated suppression of pancreatic cancer, a deadly cancer with a mere 6% 5-year survival rate and which is typically associated with p53 mutation. Our proposed work builds on initial studies of a unique panel of p53 transcriptional activation domain (TAD) mutant knock-in mice that we generated to define the downstream p53 transcriptional targets most essential for suppressing cancer development. A particularly powerful mutant is the TAD1 mutant, known as p5325,26, which is severely compromised for activation of most known p53 target genes yet still efficiently transactivates a small set of novel p53-dependent genes in fibroblasts and still retains full activity in suppressing a variety of cancers. In addition, the TD2 mutant, p5353,54 can hyperactivate a subset of p53 target genes and behaves as a super-tumor suppressor. The p53 tumor suppression associated target genes (TSAGs) activated by the p5325,26 and p5353,54 mutants largely represent novel p53 targets, which we hypothesize are critical for tumor suppression and therefore have great potential to expand our current knowledge of p53 tumor suppression mechanisms. Here, we propose to perform ChIP-seq and RNA-seq in premalignant pancreatic ductal epithelial cells expressing these mutants to identify TSAGs associated with pancreatic cancer suppression. We then propose to identify key mediators of p53 function in tumor suppression by performing CRISPR screening in mouse pancreas cancer models in vivo to identify combinations of p53 TSAGs whose loss promotes cancer. We will define the cellular functions of the proteins encoded by p53 TSAGs in different cellular processes regulated by p53, including cell-cycle progression, apoptosis, metabolism, and invasion/metastasis, using overexpression and knockdown approaches. Detailed analyses of p53 TSAG function will help elucidate cellular functions most critical for tumor suppression. We will next use mass spectrometry and siRNA screening to identify the co-factors through which the TADs act to induce p53 TSAGs and to suppress pancreatic cancer, providing another level of understanding of tumor suppression. Finally, we will leverage our expertise in studying p53 TADs to define the importance of TADs for p53 GOF activity. We will use a systematic approach to analyze the TADs at the genetic, genomic and biochemical levels to understand how tumor-derived p53 GOF mutants exert their effects. Collectively, these studies will provide crucial insight into how to modulate p53 pathways during therapeutic strategies for cancer.